CN101372512B - Anticoagulated blood polypeptides and uses thereof - Google Patents
Anticoagulated blood polypeptides and uses thereof Download PDFInfo
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- CN101372512B CN101372512B CN2007101206853A CN200710120685A CN101372512B CN 101372512 B CN101372512 B CN 101372512B CN 2007101206853 A CN2007101206853 A CN 2007101206853A CN 200710120685 A CN200710120685 A CN 200710120685A CN 101372512 B CN101372512 B CN 101372512B
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Abstract
The invention discloses an anticoagulant polypeptide, belonging to the field of biomedicine. The primary structure of the anticoagulant polypeptide is shown as the general formula: (D)-FPRP-X1-X2-X3-X4-X5-QGDFEPIPEDAYDE-NH2. The invention has the advantages that the invention designs and synthesizes one type of anticoagulant polypeptide according to the structure characteristics and action mode of thrombin and Bivalirudin which is anticoagulant drug, and the polypeptide can inhibit the activity of the thrombin efficiently and specifically; furthermore, the number of amino acid in the anticoagulant polypeptide is equal to that of the Bivalirudin, thus synthesis is easy, and the activity of anticoagulation is stronger.
Description
Technical field
The present invention relates to anticoagulated blood polypeptides and uses thereof, belong to biomedicine field.
Background technology
Blood coagulation is the important physiology defence process of body, but pathologic thrombus serious harm human health.The main anticoagulation medicine of domestic and international clinical use at present is unfractionated heparin, low molecular weight heparin, warfarin etc., and these medicines exist and cause thrombopenia, side effect or the close shortcomings such as laboratory monitoring of needs such as hemorrhage.And polypeptide class anticoagulation medicine as r-hirudin, Bivalirudin and small molecules class peptide medicine (as Xi Meijia group etc.) that can be oral etc., has outstanding advantages such as rapid-action, that side effect is less, be from now on anticoagulant give priority to direction.
Zymoplasm plays central role in the thrombus process: (1) cracking Fibrinogen becomes scleroproein; (2) incitant V, VIII and XI, these factors form more zymoplasm conversely; (3) stimulating platelet.In addition, make the crosslinked stable embolism of scleroproein by incitant VIII.Zymoplasm has three lands: scleroproein land, substrate catalysis (activity) district and heparin land.Zymoplasm is by containing 36 amino acid whose A chains and 259 amino acid whose B chains are formed, the A chain is connected by disulfide linkage with the B chain, as other serine proteases, it comprises a typical catalysis triplet (Asp102, His57 and Ser195), its S1 land resembles a dark pocket, the Asp189 of bottom can form hydrogen bond or electrostatic interaction with the amino or the guanidine radicals of substrate, and the hydrophobic region in this district can be in conjunction with hydrophobic grouping.Different with other trypsinase is to also have second hydrophobic region (S2), and hydrophobic YPPW ring is contained in this district.In addition, tart Glu192 residue is in the ingress of pocket, and is very important to identification substrate and inhibitor.The scleroproein of zymoplasm and heparin land mainly are made up of several arginine and Methionin, can combine with electronegativity amino acid and group.
R-hirudin is the direct inhibitor of finding the earliest of polypeptide Thrombin-like enzyme, be made up of 65 amino-acid residues, occurring in nature has multiple r-hirudin varient, and anticoagulating active is stronger mainly contains (Salzet M.CurrPharm Des such as HV1, HV2 and HV3,2002,8 (7): 493～503).But the substrate catalytic domain of r-hirudin bind thrombin and scleroproein land, form 1: 1 mixture (binding constant Ki is up to 20fM) (Krsttenansky J L with zymoplasm, et al.FEBS Lett, 1990,269:425～429), stop zymoplasm that fibrinogen degradation is become fibrin monomer, thereby suppress condensing of fibrin monomer.R-hirudin has can not be oral, easy shortcoming such as degraded in the body, can overcome by structural modification.Can obtain the long transformation period as the r-hirudin of modifying with lipophilic compounds such as PEG molecule or stearic acid, oleic acid.RGD merges r-hirudin and then also has the platelet aggregation-against function.The genetically engineered bi-functional leech essence (RGD-hirudin) of injection has obtained State Food and Drug Administration's approval and has entered clinical study in 2005, clinical indication is the anti-freezing of vascular anastomosis postoperative, anti-bolt treatment, and can be used for treating diseases such as stenocardia, deep venous thrombosis.
The small-molecular peptides that is derived by r-hirudin is a lot, a bind thrombin catalytic domain that has, and the scleroproein land of a bind thrombin that has, active undesirable, though it is the activity that has is higher, synthetic difficult.Bivalirudin (Bivalirudin, hirulog-1) be the hirudin derivative of synthetic, contain 20 amino-acid residues, its aminoterminal can bind thrombin the substrate catalytic domain, it is scleroproein land (Bourdon P, et that the tart carboxyl terminal then can bind thrombin, al.FEBS Lett, 1991,293:163～166), the middle connection portion of forming a flexibility by 4 Gly.Bivalirudin have wideer treatment window and security than leech, but shortcoming be the both can not be oral, easily degraded in the body, the transformation period is shorter.
Small molecules class peptide antithrombotics mainly contains argatroban, Xi Meijia group, dabigatran etc.Argatroban is formed by N-α-benzenesulfonyl-L-arginine methyl esters transformation, can be reversible, bind thrombin catalytic site (Preville P, et al.Bioorg Med Chem Lett, 1997,7 (12): 1563～1566) optionally.In recent years, some argatroban analogues be synthesized (Salvagnini C, et al.Eur J Med Chem.2007,42 (1): 37-53.), as its alkaline guanidine radicals is replaced to lipophilic side chain, to improve its cell permeability and oral availability.Melagatran is the analogue of D-Phe-Pro-Arg tripeptide sequence, wherein the benzene carbon amidine base replaces arginine, the azetidine-2-carboxylic acid replaces proline(Pro), D-type Cyclohexylglycine replaces the D-phenylalanine, Melagatran is the catalytic site (GustafssonD of Trombin inhibiting reversibly, et.al.J Intern Med.2003,254 (4): 322～334).Become ester group on the carboxyl with Melagatran, amidino groups adds the amidoxime base, can be converted into oral prodrug-Xi Meijia group (Boos C J, et.al.Eur J Intern Med, 2005,16 (4): 267～278), Xi Meijia group is after heparin develops more than 50 year, first that occurs can be oral anticoagulant, it is subjected to the interference of food and other drug little, and drug effect can be predicted, does not need the anticoagulation monitoring, but because its liver toxicity, in February, 2006, Astrazeneca company announced Xi Meijia group (ExantaTM) is recalled from the market.Dabigatran (dabigatran, BIBR 953) comprise trisubstituted benzoglyoxaline core skeleton, the phenylalanine analogues that contains a 4-amino, but bind thrombin catalytic site (Bates S M, et.al.Br J Pharmacol.2005,144 (8): 1017～1028), be transformed into after the dabigatran esterification can be oral BIBR 1048 (dabigatranexexilate).Entered the III clinical trial phase at present.
The ideal anticoagulation medicine should have following characteristic: anticoagulating active is good, and drug effect can be predicted, does not need the laboratory monitoring, can be oral, and rapid-action, side effect is little etc.Present anti-freezing medicine also fails to satisfy these requirements.The primary study direction that little peptide and small molecules class peptide that can be oral or compound will be anticoagulation medicine from now on, and small molecules class peptide thrombin inhibitors has been obtained remarkable progress reduces side effect and is striving direction from now on.
Summary of the invention
The technical problem to be solved in the present invention is: provide a class novel anticoagulant peptides, such polypeptide is compared with Bivalirudin, and its anticoagulant active is stronger.
Another technical problem that the present invention will solve is: the purposes of this class anticoagulant peptides at pharmacy field is provided.
For achieving the above object, the present invention is by the following technical solutions:
Anticoagulant peptides, primary structure is shown in general formula:
(D)-FPRP-X
1-X
2-X
3-X
4-X
5-QGDFEPIPEDAYDE-NH2
Wherein:
X1 is selected among G, AEEA, K (steric), the R any one;
X2 is selected among G, K (steric), the R any one or vacancy;
X3 is selected among G, K (steric), the D any one or vacancy:
X4 is selected among G, K (steric), W, the D any one;
X5 is selected among P, the S any one or vacancy;
Wherein A is a L-Ala; D is an aspartic acid; E is a L-glutamic acid; F is a phenylalanine; (D)-F is a D type phenylalanine; G is a glycine; I is an Isoleucine; K (steric) is for having the Methionin of stearic acid side chain; P is a proline(Pro); Q is a glutamine; R is an arginine; S is a Serine; W is a tryptophane; Y is a tyrosine, and AEEA is 2-(2-(2-amino ethoxy) oxyethyl group) acetate.
The general formula I polypeptide is respectively following polypeptide, and their sequence is as follows respectively:
Polypeptide 1:(D)-FPRP-GGGG-QGDFEPIPEDAYDE-NH2
Polypeptide 2:(D)-FPRP-AEEA-G-QGDFEPIPEDAYDE-NH2
Polypeptide 3:(D)-FPRP-G-K (steric) GG-QGDFEPIPEDAYDE-NH2
Polypeptide 4:(D)-FPRP-GG-K (steric) G-QGDFEPIPEDAYDE-NH2
Polypeptide 5:(D)-FPRP-GGG-K (steric)-QGDFEPIPEDAYDE-NH2
Polypeptide 6:(D)-FPRP-GRGDS-QGDFEPIPEDAYDE-NH2
Polypeptide 7:(D)-FPRP-RGDWP-QGDFEPIPEDAYDE-NH2
Polypeptide 8:(D)-FPRP-K (steric) GGG-QGDFEPIPEDAYDE-NH2
In aforementioned polypeptides, what anticoagulating active was good especially is polypeptide 1,2,3 and 6.In addition, we have also synthesized following polypeptide, and their sequence is as follows:
Polypeptide 9:(D)-FPRP-AEEA-G-NGDFEEIPEEYL
Polypeptide 10:(D)-FPRP-PABA-G-NGDFEEIPEEYL
Polypeptide 11:(D)-FPRP-PAMBA-G-NGDFEEIPEEYL
Polypeptide 12:(D)-FPRP-GRGDWP-DFEEIPEEYL
Polypeptide 13:(D)-FPRP-RGDWPG-DFEEIPEEYL
PABA is a para-amino benzoic acid, and PAMBA is a paraaminomethyl benzoic acid.
Polypeptide provided by the present invention is the substrate catalytic domain and the scleroproein land of bind thrombin simultaneously, and has stronger anticoagulant active.Its aminoterminal is hydrophobic (D)-FPRP sequence, the substrate catalytic domain of energy bind thrombin, and zymoplasm can slowly cut amido linkage (the Witting J I between the R-P after combining with it, et.al.Biochem J, 1992,283:737～743), make its inactivation.Its carboxyl terminal aminoacid sequence integral body is electronegativity, the scleroproein land of energy bind thrombin, we have replaced 12 amino-acid residues (NGDFEEIPEEYL) of the carboxyl terminal of hirulog-1 with the carboxyl terminal sequence (QGDFEPIPEDAYDE) of r-hirudin mutation III, carboxyl terminal after the replacement has stronger electronegativity, and is stronger with the avidity of zymoplasm.Then couple together between carboxyl terminal and the aminoterminal with flexible aminoacid sequence, we have replaced one or several amino acid in the flexible sequence with artificial synthetic organic compound, in addition, introduced the Methionin that has the stearic acid side chain eventually in sequence, in the hope of slowing down the degraded of zymoplasm, strengthen anticoagulating active to polypeptide.
Methods such as the present invention can synthesize by solid phase synthesis, liquid phase, engineering bacterium expression are produced.For example, adopt solid phase synthesis, coupling amino acid sequence one by one on resin is to form the present invention; Synthetic or extraction can be expressed dna sequence dna of the present invention, be connected on a certain carrier, and transfection is in eucaryon or procaryotic cell, expresses the protein or the polypeptide that contain peptide sequence provided by the present invention, through extracting and purifying obtains polypeptide of the present invention.
Polypeptide provided by the invention can synthesize on the ABI433 type solid phase synthetic instrument of U.S.'s application system biology, and the modification of polypeptide is manual to be finished.Should protect (U.S. Advanced Chemtech company product) with Fmoc by the synthetic amino acid that uses, the resin that uses is Rink resin or Wang resin (U.S. Advanced Chemtech company product).Use I-hydroxybenzotriazole (HoBt) (U.S. Advanced Chemtech company product) to be dissolved in the N-Methyl pyrrolidone (NMP) (PE company) when synthetic as activator; use dicyclohexylcarbodiimide (DCC) (Acros company) as coupling agent, use piperidines (Piperidine) (Shanghai gill biochemistry) to remove protecting group.Amino acid all has L-type chemical structure (except the Fmoc-D-Phe-OH), and they are coupled on Rink or the Wang resin successively.
The usage quantity of resin and Fmoc protect amino acid whose usage quantity to be undertaken by 1: 5; protection amino acid (U.S. Advanced Chemtech company product) is as follows: Fmoc-D-Phe-OH; Fmoc-Pro-OH; Fmoc-Arg (Pbf)-OH; Fmoc-Gly-OH; Fmoc-Asn (Trt)-OH; Fmoc-Asp (OtBu)-OH; Fmoc-Phe-OH; Fmoc-Glu (OtBu)-OH; Fmoc-Ile-OH; Fmoc-Tyr (tBu)-OH; Fmoc-Leu-OH; Fmoc-Gln (Trt)-OH; Fmoc-Ala-OH; and the artificial reconstructed Fmoc-Lys that forms (steric)-OH; Fmoc-AEEA-OH; Fmoc-PABA and Fmoc-PAMBA are synthetic for this laboratory, and amino acid whose coupling is carried out according to the instrumentation rules.
Peptide resin after above-mentioned synthesizing (is formed: DTT (DTT) 0.5g with lysate, water 0.5ml, trifluoroacetic acid (TFA) 8.8ml, and tri isopropyl silane (TIPS) 0.2ml) cracking 2.5～3.0 hours, filter, after filtrate boils off most of trifluoroacetic acid with Rotary Evaporators, anhydrous diethyl ether with precooling precipitates, and filters to obtain peptide just, with deionized water or weak ammonia dissolved solids, filter the filtrate freeze-drying.
The thick peptide of above-mentioned freeze-drying carries out purifying with reversed-phase HPLC, and purification column is anti-phase C
18(9.4mm * 25cm), gradient eluent is acetonitrile (containing the 0.1%TFA)/water (containing 0.1%TFA) that contains different gradients to semipreparative column for Zorbax, 300SB-C18, collects target peak, and rotary evaporation is removed most of acetonitrile, and freeze-drying obtains pure peptide.
The purposes that anticoagulated blood polypeptides is used to prepare prevention and treats the medicine of thrombotic diseases.
The prevention and the treatment of deep venous thrombosis after polypeptide of the present invention can be used to perform the operation, the anticoagulant therapy that is used for the functions in patients with unstable angina of row percutaneous tranluminal coronary angioplasty (PTCA), perhaps unite the anticoagulant therapy that is used for getting involved the patient of (PCI) treatment, and alternative heparin need to be used to the patient of the thrombocytopenia that the heparin of anticoagulant therapy causes through the skin coronary artery with platelet glycoprotein II b/IIIa inhibitor.
Can will contain polypeptide of the present invention or its medicine that blocks thing, derivative and composition directly gives patient, perhaps with after suitable carrier or excipient mix give patient.The solid support material here comprises: the water-soluble carrier material, and as polyoxyethylene glycol, polyvinylpyrrolidone, organic acid etc.; The insoluble solid support material, as ethyl cellulose, cholesterol ester stearic acid etc.; The enteric solubility solid support material is as cellulose acetate phthalate and carboxylic first and second Mierocrystalline celluloses etc.Use these materials, can make following formulation: tablet, suppository, solution, capsule, aerosol, effervescent tablet and drops etc.
Use above-mentioned formulation, polypeptide that provides of the present invention and derivative thereof, block thing and analogue and composition can: administrated by injection comprises intravenous injection, subcutaneous injection, intracavitary administration etc.; Mucosa delivery as intranasal administration, plays a role in local onset or through the mucosal absorption whole body; Cavity/canal drug administration, as the per rectum administration, local onset or play a role through absorbing whole body.Above-mentioned route of administration is preferably through intravenous administration.
Advantage of the present invention is: the present invention is according to the constructional feature and the mode of action of zymoplasm and anticoagulant Bivalirudin, the synthetic anticoagulated blood polypeptides of knowing clearly of design, they can not only efficient, special anticoagulant enzymic activity, and amino acid number and Bivalirudin that it had are suitable, easily synthetic, anticoagulating active is stronger.
The invention will be further described below in conjunction with embodiment; it is not limitation of the invention; according to prior art well known in the art; embodiments of the present invention are not limited to this; therefore all this areas of having done according to the disclosure of invention be equal to replacement, all belong to protection scope of the present invention.
Description of drawings
Fig. 1 is that the HPLC behind polypeptide 1 purifying analyzes collection of illustrative plates.
Fig. 2 is that the HPLC behind polypeptide 2 purifying analyzes collection of illustrative plates.
Fig. 3 is that the HPLC behind polypeptide 3 purifying analyzes collection of illustrative plates.
Fig. 4 is that the HPLC behind polypeptide 6 purifying analyzes collection of illustrative plates.
Fig. 5 is that the HPLC behind polypeptide 7 purifying analyzes collection of illustrative plates.
Embodiment
Embodiment 1: the synthetic and purifying of polypeptide 1
One. synthetic:
Take by weighing 0.17gRink resin (0.1mmol; replacement rate 0.6g/mmol); it is 0.5mmol that each Fmoc protects amino acid whose consumption; with I-hydroxybenzotriazole (HoBt) and dicyclohexylcarbodiimide (DCC, Acros company) is condensing agent, piperidines (Piperidine; Shanghai gill biochemistry) deprotection; press the operation instructions of the ABI433A type solid phase synthetic instrument of U.S.'s application system biology, proper extension coupling time (60-90min) and deprotection time (20-30min), synthetic peptide-resin.
Get above-mentioned peptide-resin, putting into the 10ml lysate (forms: 0.5g DTT (DTT), 0.5ml water, 8.8ml trifluoroacetic acid (TFA), 0.2ml tri isopropyl silane (TIPS)), cracking 3.0 hours, filter with G3 glass sand core funnel, boil off most of filtrate to the about 2ml of residual liquid with Rotary Evaporators, precipitate, left standstill 1 hour with the anhydrous diethyl ether of 100ml precooling, G4 glass sand core funnel filters and obtains peptide just, with 1% weak ammonia dissolved solids, the filtrate freeze-drying gets thick peptide, and purity is about 60%.
Two. purifying:
Get thick peptide 39.0mg HPLC purifying, chromatographic column is anti-phase C
18Semipreparative column (Zorbax, 300SB-C18,9.4mm * 25cm).Moving phase: A, acetonitrile (containing 0.1%TFA); B, water (containing 0.1%TFA).Gradient is: 1-25min, and 15%-70%A, flow velocity 3ml/min, UV214nm detects, and goes up sample 5mg at every turn.Collect target components, rotary evaporation is removed most of acetonitrile, and freeze-drying obtains pure peptide 19.2mg.The purity check of polypeptide 1 is seen Fig. 1.
HPLC behind the peptide purification analyzes collection of illustrative plates:
Flow velocity: 1ml/min
Detect wavelength: 214nm
Column temperature: 25 ℃
Gradient: gradient is
0-1min，0-15％A
1-25min，15％-70％A
25-27min，80％-90％A
A, acetonitrile (containing 0.1%TFA); B, water (containing 0.1%TFA)
Applied sample amount: 50 μ g
Analytical column: Kromasil, C-18 post (Beijing Analytical Instrument Factory), 5 μ m, Φ 4.6mm * 250mm
Embodiment 2: the synthetic and purifying of polypeptide 2
One. synthetic:
Take by weighing 0.17gRink resin (0.1mmol; replacement rate 0.6g/mmol); it is 0.5mmol that each Fmoc protects amino acid whose consumption; with I-hydroxybenzotriazole (HoBt) and dicyclohexylcarbodiimide (DCC, Acros company) is condensing agent, piperidines (Piperidine; Shanghai gill biochemistry) deprotection; press the operation instructions of the ABI433A type solid phase synthetic instrument of U.S.'s application system biology, proper extension coupling time (60-90min) and deprotection time (20-30min), synthetic peptide-resin.
Get above-mentioned peptide-resin, putting into the 10ml lysate (forms: 0.5g DTT (DTT), 0.5ml water, 8.8ml trifluoroacetic acid (TFA), 0.2ml tri isopropyl silane (TIPS)), cracking 3.0 hours, filter with G3 glass sand core funnel, boil off most of filtrate to the about 2ml of residual liquid with Rotary Evaporators, precipitate, left standstill 1 hour with the anhydrous diethyl ether of 100ml precooling, G4 glass sand core funnel filters and obtains peptide just, with 1% weak ammonia dissolved solids, the filtrate freeze-drying gets thick peptide, and purity is about 65%.
Two. purifying:
Get thick peptide 39.0mg HPLC purifying, chromatographic column is anti-phase C
18Semipreparative column (Zorbax, 300SB-C18,9.4mm * 25cm).Moving phase: A, acetonitrile (containing 0.1%TFA); B, water (containing 0.1%TFA).Gradient is: 1-25min, and 15%-70%A, flow velocity 3ml/min, UV214nm detects, and goes up sample 5mg at every turn.Collect target components, rotary evaporation is removed most of acetonitrile, and freeze-drying obtains pure peptide 21.8mg.The purity check of polypeptide 2 is seen Fig. 2.
HPLC behind the peptide purification analyzes collection of illustrative plates:
Flow velocity: 1ml/min
Detect wavelength: 214nm
Column temperature: 25 ℃
Gradient: gradient is
0-1min，0-15％A
1-25min，15％-70％A
25-27min，80％-90％A
A, acetonitrile (containing 0.1%TFA); B, water (containing 0.1%TFA)
Applied sample amount: 50 μ g
Analytical column: Kromasil, C-18 post (Beijing Analytical Instrument Factory), 5 μ m, Φ 4.6mm * 250mm
Embodiment 3: the synthetic and purifying of polypeptide 3
One. synthetic:
Take by weighing 0.17gRink resin (0.1mmol; replacement rate 0.6g/mmol); it is 0.5mmol that each Fmoc protects amino acid whose consumption; with I-hydroxybenzotriazole (HoBt) and dicyclohexylcarbodiimide (DCC, Acros company) is condensing agent, piperidines (Piperidine; Shanghai gill biochemistry) deprotection; press the operation instructions of the ABI433A type solid phase synthetic instrument of U.S.'s application system biology, proper extension coupling time (60-90min) and deprotection time (20-30min), synthetic peptide-resin.
Get above-mentioned peptide-resin, putting into the 10ml lysate (forms: 0.5g DTT (DTT), 0.5ml water, 8.8ml trifluoroacetic acid (TFA), 0.2ml tri isopropyl silane (TIPS)), cracking 3.0 hours, filter with G3 glass sand core funnel, boil off most of filtrate to the about 2ml of residual liquid with Rotary Evaporators, precipitate, left standstill 1 hour with the anhydrous diethyl ether of 100ml precooling, G4 glass sand core funnel filters and obtains peptide just, with 1% weak ammonia dissolved solids, the filtrate freeze-drying gets thick peptide, and purity is about 55%.
Two. purifying:
Get thick peptide 38.0mg HPLC purifying, chromatographic column is anti-phase C
18Semipreparative column (Zorbax, 300SB-C18,9.4mm * 25cm).Moving phase: A, acetonitrile (containing 0.1%TFA); B, water (containing 0.1%TFA).Gradient is: 1-25min, and 15%-70%A, flow velocity 3ml/min, UV214nm detects, and goes up sample 5mg at every turn.Collect target components, rotary evaporation is removed most of acetonitrile, and freeze-drying obtains pure peptide 14.3mg.The purity check of polypeptide 3 is seen Fig. 3.
HPLC behind the peptide purification analyzes collection of illustrative plates:
Flow velocity: 1ml/min
Detect wavelength: 214nm
Column temperature: 25 ℃
Gradient: gradient is:
0-1min，0-45％A
1-25min，45％-80％A
25-27min，80％-90％A
A, acetonitrile (containing 0.1%TFA); B, water (containing 0.1%TFA)
Applied sample amount: 50 μ g
Analytical column: Kromasil, C-18 post (Beijing Analytical Instrument Factory), 5 μ m, Φ 4.6mm * 250mm
Embodiment 4: the synthetic and purifying of polypeptide 6
One. synthetic:
Take by weighing 0.17gRink resin (0.1mmol; replacement rate 0.6g/mmol); it is 0.5mmol that each Fmoc protects amino acid whose consumption; with I-hydroxybenzotriazole (HoBt) and dicyclohexylcarbodiimide (DCC, Acros company) is condensing agent, piperidines (Piperidine; Shanghai gill biochemistry) deprotection; press the operation instructions of the ABI433A type solid phase synthetic instrument of U.S.'s application system biology, proper extension coupling time (60-90min) and deprotection time (20-30min), synthetic peptide-resin.
Get above-mentioned peptide-resin, putting into the 10ml lysate (forms: 0.5g DTT (DTT), 0.5ml water, 8.8ml trifluoroacetic acid (TFA), 0.2ml tri isopropyl silane (TIPS)), cracking 3.0 hours, filter with G3 glass sand core funnel, boil off most of filtrate to the about 2ml of residual liquid with Rotary Evaporators, precipitate, left standstill 1 hour with the anhydrous diethyl ether of 100ml precooling, G4 glass sand core funnel filters and obtains peptide just, with 1% weak ammonia dissolved solids, the filtrate freeze-drying gets thick peptide, and purity is about 65%.
Two. purifying:
Get thick peptide 35.0mg HPLC purifying, chromatographic column is anti-phase C
18Semipreparative column (Zorbax, 300SB-C18,9.4mm * 25cm).Moving phase: A, acetonitrile (containing 0.1%TFA); B, water (containing 0.1%TFA).Gradient is: 1-25min, and 15%-70%A, flow velocity 3ml/min, UV214nm detects, and goes up sample 5mg at every turn.Collect target components, rotary evaporation is removed most of acetonitrile, and freeze-drying obtains pure peptide 16.5mg.The purity check of polypeptide 6 is seen Fig. 4.
HPLC behind the peptide purification analyzes collection of illustrative plates:
Flow velocity: 1ml/min
Detect wavelength: 214nm
Column temperature: 25 ℃
Gradient: gradient is
0-1min，0-15％A
1-25min，15％-70％A
25-27min，80％-90％A
A, acetonitrile (containing 0.1%TFA); B, water (containing 0.1%TFA)
Applied sample amount: 50 μ g
Analytical column: Kromasil, C-18 post (Beijing Analytical Instrument Factory), 5 μ m, Φ 4.6mm * 250mm
Embodiment 5: the synthetic and purifying of polypeptide 7
One. synthetic:
Take by weighing 0.17gRink resin (0.1mmol; replacement rate 0.6g/mmol); it is 0.5mmol that each Fmoc protects amino acid whose consumption; with I-hydroxybenzotriazole (HoBt) and dicyclohexylcarbodiimide (DCC, Acros company) is condensing agent, piperidines (Piperidine; Shanghai gill biochemistry) deprotection; press the operation instructions of the ABI433A type solid phase synthetic instrument of U.S.'s application system biology, proper extension coupling time (60-90min) and deprotection time (20-30min), synthetic peptide-resin.
Get above-mentioned peptide-resin, putting into the 10ml lysate (forms: 0.5g DTT (DTT), 0.5ml water, 8.8ml trifluoroacetic acid (TFA), 0.2ml tri isopropyl silane (TIPS)), cracking 3.0 hours, filter with G3 glass sand core funnel, boil off most of filtrate to the about 2ml of residual liquid with Rotary Evaporators, precipitate, left standstill 1 hour with the anhydrous diethyl ether of 100ml precooling, G4 glass sand core funnel filters and obtains peptide just, with 1% weak ammonia dissolved solids, the filtrate freeze-drying gets thick peptide, and purity is about 60%.
Two. purifying:
Get thick peptide 40.0mg HPLC purifying, chromatographic column is anti-phase C
18Semipreparative column (Zorbax, 300SB-C18,9.4mm * 25cm).Moving phase: A, acetonitrile (containing 0.1%TFA); B, water (containing 0.1%TFA).Gradient is: 1-25min, and 15%-70%A, flow velocity 3ml/min, UV214nm detects, and goes up sample 5mg at every turn.Collect target components, rotary evaporation is removed most of acetonitrile, and freeze-drying obtains pure peptide 18.7mg.The purity check of polypeptide 7 is seen Fig. 5.
HPLC behind the peptide purification analyzes collection of illustrative plates:
Flow velocity: 1ml/min
Detect wavelength: 214nm
Column temperature: 25 ℃
Gradient: gradient is
0-1min，0-15％A
1-25min，15％-70％A
25-27min，80％-90％A
A, acetonitrile (containing 0.1%TFA); B, water (containing 0.1%TFA)
Applied sample amount: 50 μ g
Analytical column: Kromasil, C-18 post (Beijing Analytical Instrument Factory), 5 μ m, Φ 4.6mm * 250mm
Embodiment 6: the mensuration of anticoagulant active
One. experiment purpose
Measure the anticoagulant active of polypeptide provided by the present invention, estimate it compares anticoagulating active in control peptide hirulog-1 power.And measure the power of polypeptide provided by the present invention with respect to the zymoplasm anticoagulant active of different genera (pig, ox and people).
Two. experiment material and method
(1) experiment material
1. damping fluid: contain the Tris-HCl solution of the 0.05mol/L of 0.05mmol/L NaCl, PH7.4.
2.0.5% fibrinogen solution (fibrinogen solution that comprises pig, ox and people) is prepared with above-mentioned damping fluid.
3. the thrombin solution of different concns gradient (thrombin solution that comprises pig, ox and people): it (is 1 titration volume that concentration is respectively 20NIH/ml, both 1V, be 0.1ATU), 40NIH/ml (2V), 100NIH/ml (5V), 200NIH/ml (10V), 400NIH/ml (20V), 800NIH/ml (40V) and 1000NIH/ml (50V).
4. each anticoagulant peptides solution: take by weighing each polypeptide 5mg respectively, be mixed with the solution of 2.5mmol/L respectively.In the experimentation, dilute respectively according to the power of each polypeptide anticoagulating active and to be suitable concn.
5.Hirulog-1: available from the triumphant prompt biological medicine in Chengdu Science and Technology Ltd., take by weighing 5mg, be mixed with the solution of 2.5mmol/L.Redilution is a suitable concn in the experimentation.
(2) method
1. the mensuration of anticoagulant active adopts zymoplasm volumetry (Markwardt F.Methods Enzymol, 1970,69:924-932. Chen Hua friend etc., biotechnology, 2002,12 (6): 24-25).
The concrete operations step is as follows: get several small test tubes (0.75*10cm), add 200 μ L, 0.5% fibrinogen solution and 50 μ L polypeptide solution to be measured respectively, 37 ℃ of incubation 5min, getting one props up, the NIH/ml thrombin solution 5 μ L of Dropwise 5 V shake up rapidly, if solidify in the 1min, illustrate that anticoagulating active is less than 5V, then in another test tube, add the thrombin solution 5 μ L of 1V again,, add the thrombin solution 5 μ L of 1V again if do not coagulate in the 1min, so go down, till in 1min, solidifying.If testing sample does not solidify in the 1min at the thrombin solution 5 μ L that add 5V, then add the thrombin solution 5 μ L of 20V again, shake up rapidly,, illustrate active between 5V-25V if solidify in the 1min, so get a small test tube again, be added dropwise to the thrombin solution 5 μ L of 20V, shake up rapidly, if do not solidify in the 1min, then get the thrombin solution 5 μ L that add 1V in the test tube again,, add the thrombin solution 5 μ L of 1V again if do not coagulate in the 1min, so go down, till in 1min, solidifying.
2. the determination of activity process of polypeptide 3 following (polypeptide 3 concentration are 0.1mM, and zymoplasm is a pig thrombiase):
Get 5 small test tubes, add 200 μ L, 0.5% fibrinogen solution and 50 μ L polypeptide solution to be measured respectively, 37 ℃ of incubation 5min, get one and prop up, the NIH/ml thrombin solution 5 μ L of Dropwise 5 V shake up rapidly, do not solidify in the 1min, be added dropwise to the thrombin solution 5 μ L of 20V again, shake up rapidly, do not solidify in the 1min, add the thrombin solution 5 μ L of 20V again, shake up rapidly, do not coagulate yet, when adding the thrombin solution of 5 20V altogether, occur solidifying, illustrate that activity is again between the 85V-105V, then get a small test tube in addition, add the thrombin solution 5 μ L of 40V, shake up rapidly, do not solidify in the 1min, add the thrombin solution 5 μ L of 40V again, shake up rapidly, do not solidify in the 1min, the thrombin solution 5 μ L that add 20V again, shake up rapidly, do not coagulate yet, add the thrombin solution 5 μ L of 1V again, shake up rapidly, occur in the 1min solidifying, illustrate that its anticoagulant active is 101V, i.e. 10.1ATU, repeat 3 times, the result gets its mean value.
All the other polypeptide are all measured anticoagulating active as above-mentioned method.
Three. experimental result
Part of polypeptide provided by the present invention and control peptide hirulog-1 see Table 1 with respect to the anticoagulating active of different genera (pig, ox and people) zymoplasm.
Table 1: the anticoagulating active experimental result of part of polypeptide
Four. conclusion
Four anticoagulating actives are arranged apparently higher than control peptide hirulog-1 (polypeptide 1, polypeptide 2, polypeptide 3 and polypeptide 6) in the polypeptide provided by the present invention: to the anticoagulant active of pig thrombiase be control peptide 3-4 doubly, to the activity of thrombin of beef be control peptide 6-8 doubly, human thrombin is then equated or nearly 2 times.
Embodiment 7: the mensuration that suppresses constant (Ki)
One. experiment purpose
By analyzing chromogenic substrate Chromozym TH by the degraded of zymoplasm, calculate the kinetic parameter of each polypeptide, estimate its anticoagulating active.
Two. experiment material and method
(1) experiment material
1. damping fluid: 0.01M Hepes/0.01M Tris, 0.1M NaCl, 0.1%PEG6000, PH7.4.
2. substrate: Chromozym TH (Tos-Gly-Pro-Arg-PNA is available from Roche Holding Ag), being mixed with concentration respectively is 25 μ M, 33 μ M, 40 μ M, 50 μ M, 100 μ M, 125 μ M, 200 μ M, 330 μ M, 500 μ M, 1000 μ M.
3. thrombin of beef (available from sigma company) solution: be mixed with 5NIH/ml.
4. the concentration of anticoagulant peptide: be mixed with 0.05 μ M, 0.10 μ M, 0.15 μ M. respectively
(2) method
Adopt chromogenic substrate method (Cappiello M, et.al.Biochemical.Biochemical Pharmacology, 1996,52:1141-1146.Maraganore J M, et.al.Biochemistry, 1990,29:7095-7101), substrate Chromozym TH (Tos-Gly-Pro-Arg-PNA) can be produced p-Nitroaniline by the zymoplasm cutting, and the latter has very strong absorption at the 405nm wavelength, so can determine the inhibition activity of anticoagulant peptide to zymoplasm by the generation of monitoring p-Nitroaniline.
The concrete operations step is as follows: 0.05ml thrombin of beef solution and 0.05ml anticoagulant peptide solution are joined 0.35ml damping fluid (0.01M Hepes/0.01M Tris, 0.1M NaCl, 0.1%PEG6000, PH7.4) in, mixing, in 2min, add 0.05ml Chromozym TH solution (final concentration is respectively 2.5 μ M, 3.3 μ M, 4.0 μ M, 5.0 μ M, 10.0 μ M, 12.5 μ M, 20.0 μ M, 33.0 μ M, 50.0 μ M, 100.0 μ M), react 10min under the room temperature, BECKMAN DU640 spectrophotometer is monitored its absorbancy in the 405nm wavelength and is changed.Km and Vmax determine that by the double-reciprocal plot method Ki value suppresses Equation for Calculating by competitive and noncompetitive.
Three. experimental result
Part of polypeptide provided by the present invention and control peptide hirulog-1 suppress the inhibition constant K i value of thrombin of beef, see Table 2.
Table 2: the Ki value of part of polypeptide
Anticoagulant peptide Ki (nM)
Hirulog-1 21.0
Polypeptide 1 10.4
Polypeptide 3 15.2
Polypeptide 6 15.5
Polypeptide 7 15.0
Four. conclusion
The Ki value of polypeptide 1,3,6,7 is all less than hirulog-1, and this illustrates all strong than hirulog-1 of its anticoagulating active.
In sum, the invention provides the active anticoagulant peptides of a class Trombin inhibiting.The constructional feature and the mechanism of action according to zymoplasm and r-hirudin, the present invention designs and has synthesized the novel anti-freezing polypeptide of bind thrombin substrate catalytic domain and scleroproein land simultaneously, and replace natural amino acid with artificial synthetic organic compound, in sequence, introduce stearic acid side chain etc., obtain stronger, the better polypeptide of metabolism of anticoagulating active.The medicine that can prepare prevention and treatment thrombotic diseases with polypeptide of the present invention.
Sequence table
Claims (2)
1. anticoagulant peptides, primary structure is as follows:
(D)-FPRP-GK(steric)GG-QGDFEPIPEDAYDE-NH
2
Wherein A is a L-Ala; D is an aspartic acid; E is a L-glutamic acid; F is a phenylalanine; (D)-F is a D type phenylalanine; G is a glycine; I is an Isoleucine; K (steric) is for having the Methionin of stearic acid side chain; P is a proline(Pro); Q is a glutamine; R is an arginine; Y is a tyrosine.
2. the described anticoagulant peptides of claim 1 is used to prepare the purposes of preventing and treating the medicine of thrombotic diseases.
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| CN101906150B (en) * | 2010-06-28 | 2013-01-09 | 上海昂博生物技术有限公司 | Preparation method of Bivalirudin |
| CN103421110B (en) * | 2013-04-14 | 2015-04-22 | 复旦大学 | DTI (Direct thrombin inhibitor) peptides and application thereof |
| CN104231054A (en) * | 2014-09-01 | 2014-12-24 | 王跃建 | 1L-10 polypeptide inhibitor |
| CN105175510B (en) * | 2015-10-22 | 2018-06-01 | 江苏大学 | The polypeptide with anticoagulant active of display technique of bacteriophage screening |
| CN112430254B (en) * | 2020-11-25 | 2021-10-26 | 广东海洋大学 | Anticoagulant active peptide derivative and preparation method and application thereof |
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| US5516656A (en) * | 1990-11-08 | 1996-05-14 | Nippon Mining Company, Limited | Production of a new hirudin analog and anticoagulant pharmaceutical composition containing the same |
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| Title |
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| Paul Bourdon et al.Structure-function relationships of hirulog peptide interactions with thrombin.《FEBS LETTERS》.1991,第294卷(第3期), * |
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